Ceramide synthase CERS4 gene downregulation is associated with KRAS mutation in colorectal cancer.

Ceramide, the central molecule in sphingolipid synthesis, is a bioactive lipid that serves as a regulatory molecule in the anti-inflammatory responses, apoptosis, programmed necrosis, autophagy, and cell motility of cancer cells. In particular, the authors have reported differences in sphingolipid content in colorectal cancer tissues. The associations among genetic mutations, clinicopathological factors, and sphingolipid metabolism in colorectal cancer (CRC) have not been investigated. The objective of this study is to investigate the association between genes associated with sphingolipid metabolism, genetic variations in colorectal cancer (CRC), and clinicopathological factors in CRC patients. We enrolled 82 consecutive patients with stage I-IV CRC who underwent tumor resection at a single institution in 2019-2021. We measured the expression levels of genes related to sphingolipid metabolism and examined the relationships between CRC gene mutations and the clinicopathological data of each individual patient. The relationship between CRC gene mutations and expression levels of ceramide synthase (CERS), N-acylsphingosine amidohydrolase (ASAH), and alkaline ceramidase (ACER) genes involved in sphingolipid metabolism was examined CRES4 expression was significantly lower in the CRC KRAS gene mutation group (p = 0.004); vascular invasion was more common in colorectal cancer patients with high CERS4 expression (p = 0.0057). By examining the correlation between sphingolipid gene expression and clinical factors, we were able to identify cancer types in which sphingolipid metabolism is particularly relevant. CERS4 expression was significantly reduced in KRAS mutant CRC. Moreover, CRC with decreased CERS4 showed significantly more frequent venous invasion.

Cathepsin G-induced malignant progression of MCF-7 cells involves suppression of PAF signaling through induced expression of PAFAH1B2.

Breast cancer is primarily classified into ductal and lobular types, as well as into noninvasive and invasive cancer. Invasive cancer involves lymphatic and hematogenous metastasis. In breast cancer patients with distant metastases, a neutrophil-derived serine protease; cathepsin G (Cat G), is highly expressed in breast cancer cells. Cat G induces cell migration and multicellular aggregation of MCF-7 human breast cancer cells; however, the mechanism is not clear. Recently, platelet-activating factor (PAF)-acetylhydrolase (PAF-AH), the enzyme responsible for PAF degradation, was reported to be overexpressed in some tumor types, including pancreatic and breast cancers. In this study, we investigated whether PAF-AH is involved in Cat G-induced aggregation and migration of MCF-7 cells. We first showed that Cat G increased PAF-AH activity and elevated PAFAH1B2 expression in MCF-7 cells. The elevated expression of PAFAH1B2 was also observed in human breast cancer tissue specimens by immunohistochemical analysis. Furthermore, knockdown of PAFAH1B2 in MCF-7 cells suppressed the cell migration and aggregation induced by low concentrations, but not high concentrations, of Cat G. Carbamoyl PAF (cPAF), a nonhydrolyzable PAF analog, completely suppressed Cat G-induced migration of MCF-7 cells. In addition, PAF receptor (PAFR) inhibition induced cell migration of MCF-7 cells even in the absence of Cat G, suggesting that Cat G suppresses the activation of PAFR through enhanced PAF degradation due to elevated expression of PAFAH1B2 and thereby induces malignant phenotypes in MCF-7 cells. Our findings may lead to a novel therapeutic modality for treating breast cancer by modulating the activity of Cat G/PAF signaling.

Controlled Tetradeuteration of Straight-Chain Fatty Acids: Synthesis, Application, and Insight into the Metabolism of Oxidized Linoleic Acid.

We describe a concise and reliable protocol for the precisely controlled tetradeuteration of straight-chain fatty acids (FAs) at the α- and ß-positions that is generally applicable to a variety of FAs, including trans-FAs, polyunsaturated FAs (PUFAs), and their oxidized derivatives. The precisely controlled introduction of four deuterium atoms into the FAs enables their persistent and quantitative tracking by LC-MS/MS analysis based on their molecular structures. In addition, the phosphatidylcholine (PC) species prepared from the tetradeuterated FAs thus obtained give a diagnostic peak, namely, a phosphocholine fragment that contains deuterium, in the LC-MS/MS analysis. With these features, the metabolism of a representative oxidized linoleic acid, that is, hydroxyoctadecadienoic acid (HODE), was investigated, leading to the identification of acyltransferases that transfer the acyl moiety derived from HODE to lysophosphatidylcholine.

Glycosphingolipids with Very Long-Chain Fatty Acids Accumulate in Fibroblasts from Adrenoleukodystrophy Patients.

Adrenoleukodystrophy (X-ALD) is an X-linked genetic disorder caused by mutation of the ATP-binding cassette subfamily D member 1 gene, which encodes the peroxisomal membrane protein, adrenoleukodystrophy protein (ALDP). ALDP is associated with the transport of very-long-chain fatty acids (VLCFAs; carbon chain length ≥ 24) into peroxisomes. Defective ALDP leads to the accumulation of saturated VLCFAs in plasma and tissues, which results in damage to myelin and the adrenal glands. Here, we profiled the glycosphingolipid (GSL) species in fibroblasts from X-ALD patients. Quantitative analysis was performed using liquid chromatography-electrospray ionization-tandem mass spectrometry with a chiral column in multiple reaction monitoring (MRM) mode. MRM transitions were designed to scan for precursor ions of long-chain bases to detect GSLs, neutral loss of hexose to detect hexosylceramide (HexCer), and precursor ions of phosphorylcholine to detect sphingomyelin (SM). Our results reveal that levels of C25 and C26-containing HexCer, Hex2Cer, NeuAc-Hex2Cer, NeuAc-HexNAc-Hex2Cer, Hex3Cer, HexNAc-Hex3Cer, and SM were elevated in fibroblasts from X-ALD patients. In conclusion, we precisely quantified SM and various GSLs in fibroblasts from X-ALD patients and determined structural information of the elevated VLCFA-containing GSLs.

N-(4-Hydroxyphenyl) Retinamide Suppresses SARS-CoV-2 Spike Protein-Mediated Cell-Cell Fusion by a Dihydroceramide Δ4-Desaturase 1-Independent Mechanism.

The membrane fusion between the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and host cells is essential for the initial step of infection; therefore, the host cell membrane components, including sphingolipids, influence the viral infection. We assessed several inhibitors of the enzymes pertaining to sphingolipid metabolism, against SARS-CoV-2 spike protein (S)-mediated cell-cell fusion and viral infection. N-(4-Hydroxyphenyl) retinamide (4-HPR), an inhibitor of dihydroceramide Δ4-desaturase 1 (DES1), suppressed cell-cell fusion and viral infection. The analysis of sphingolipid levels revealed that the inhibition efficiencies of cell-cell fusion and viral infection in 4-HPR-treated cells were consistent with an increased ratio of saturated sphinganine-based lipids to total sphingolipids. We investigated the relationship of DES1 with the inhibition efficiencies of cell-cell fusion. The changes in the sphingolipid profile induced by 4-HPR were mitigated by the supplementation with exogenous cell-permeative ceramide; however, the reduced cell-cell fusion could not be reversed. The efficiency of cell-cell fusion in DES1 knockout (KO) cells was at a level comparable to that in wild-type (WT) cells; however, the ratio of saturated sphinganine-based lipids to the total sphingolipids was higher in DES1 KO cells than in WT cells. 4-HPR reduced cell membrane fluidity without any significant effects on the expression or localization of angiotensin-converting enzyme 2, the SARS-CoV-2 receptor. Therefore, 4-HPR suppresses SARS-CoV-2 S-mediated membrane fusion through a DES1-independent mechanism, and this decrease in membrane fluidity induced by 4-HPR could be the major cause for the inhibition of SARS-CoV-2 infection. IMPORTANCE Sphingolipids could play an important role in SARS-CoV-2 S-mediated membrane fusion with host cells. We studied the cell-cell fusion using SARS-CoV-2 S-expressing cells and sphingolipid-manipulated target cells, with an inhibitor of the sphingolipid metabolism. 4-HPR (also known as fenretinide) is an inhibitor of DES1, and it exhibits antitumor activity and suppresses cell-cell fusion and viral infection. 4-HPR suppresses membrane fusion through a decrease in membrane fluidity, which could possibly be the cause for the inhibition of SARS-CoV-2 infection. There is accumulating clinical data on the safety of 4-HPR. Therefore, it could be a potential candidate drug against COVID-19.

Development of a liquid chromatography-electrospray ionization tandem mass spectrometric method for the simultaneous analysis of free fatty acids.

Fatty acids (FAs) play important roles in several physiological and pathophysiological processes, functioning as both nonesterified free FAs (FFAs) and components of other lipid classes. Although many lipid classes are readily measured using liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS), the measurement of FFAs by this method is not straightforward because of inconsistent fragmentation behaviours. In this study, we describe a strategy to measure FFAs using conventional reverse-phase LC-ESI-MS/MS, without derivatization. The strategy combines three key methods: (i) an isocratic LC separation with a high organic solvent ratio, (ii) postcolumn base addition, and (iii) pseudo-multiple reaction monitoring. The method facilitates the measurement of ultra-long-chain FAs, the accumulation of which is a common biochemical abnormality in peroxisomal disorders. This study delivers a broad strategy that measures a wide spectrum of FFA species in complex biological samples.

Mycobacterium leprae promotes triacylglycerol de novo synthesis through induction of GPAT3 expression in human premonocytic THP-1 cells.

Mycobacterium leprae (M. leprae) is the etiological agent of leprosy, and the skin lesions of lepromatous leprosy are filled with numerous foamy or xanthomatous histiocytes that are parasitized by M. leprae. Lipids are an important nutrient for the intracellular survival of M. leprae. In this study, we attempted to determine the intracellular lipid composition and underlying mechanisms for changes in host cell lipid metabolism induced by M. leprae infection. Using high-performance thin-layer chromatography (HPTLC), we demonstrated specific induction of triacylglycerol (TAG) production in human macrophage THP-1 cells following M. leprae infection. We then used [14C] stearic acid tracing to show incorporation of this newly synthesized host cell TAG into M. leprae. In parallel with TAG accumulation, expression of host glycerol-3-phosphate acyltransferase 3 (GPAT3), a key enzyme in de novo TAG synthesis, was significantly increased in M. leprae-infected cells. CRISPR/Cas9 genome editing of GPAT3 in THP-1 cells (GPAT3 KO) dramatically reduced accumulation of TAG following M. leprae infection, intracellular mycobacterial load, and bacteria viability. These results together suggest that M. leprae induces host GPAT3 expression to facilitate TAG accumulation within macrophages to maintain a suitable environment that is crucial for intracellular survival of these bacilli.

Very long-chain fatty acids are accumulated in triacylglycerol and nonesterified forms in colorectal cancer tissues.

Colorectal cancer (CRC) is a major cancer, and its precise diagnosis is especially important for the development of effective therapeutics. In a series of metabolome analyses, the levels of very long chain fatty acids (VLCFA) were shown to be elevated in CRC tissues, although the endogenous form of VLCFA has not been fully elucidated. In this study we analyzed the amount of nonesterified fatty acids, acyl-CoA species, phospholipids and neutral lipids such as cholesterylesters using liquid-chromatography-mass spectrometry. Here we showed that VLCFA were accumulated in triacylglycerol (TAG) and nonesterified forms in CRC tissues. The levels of TAG species harboring a VLCFA moiety (VLCFA-TAG) were significantly correlated with that of nonesterified VLCFA. We also showed that the expression level of elongation of very long-chain fatty acids protein 1 (ELOVL1) is increased in CRC tissues, and the inhibition of ELOVL1 decreased the levels of VLCFA-TAG and nonesterified VLCFA in CRC cell lines. Our results suggest that the upregulation of ELOVL1 contributes to the accumulation of VLCFA-TAG and nonesterified VLCFA in CRC tissues.

Mass spectrometry in combination with a chiral column and multichannel-MRM allows comprehensive analysis of glycosphingolipid molecular species from mouse brain.

Glycosphingolipids (GSLs) exist exclusively in the outer leaflet of plasma membrane in mammalian cells and have diverse structures including different classes of sugars and various molecular species of ceramide moieties. Establishing methods that measure each molecular species in GSL classes should aid functional characterization of GSLs and reveal details about the mechanism of pathogenesis in glycosphingolipidoses. Using an IF-3 chiral column that has never been used for lipid analyses, we developed a liquid chromatography-mass spectrometry (LC-MS) method to separate various GSLs based on sugar and ceramide moieties. To examine GSLs in detail a multichannel-multiple reaction monitoring (multichannel-MRM) mode was used and covered a range of 500-2000 Da. Common fragment ions detected with higher collision energy in the positive ion mode were m/z 264 and 292, and are derived from d18:1 and d20:1 ions, respectively. Both species were used as product ions in the multichannel-MRM for the simultaneous measurement of neutral GSLs, gangliosides and sulfatides. Comprehensive analysis of GSLs in mouse brain using this method revealed that for gangliosides and LacCer, d18:1-C18:0 and d20:1-C18:0 were the major molecular species, whereas d18:1-C24:0 and d18:1-C24:1 were the major molecular species of sulfatides. The results revealed a diverse GSL fatty acid profile. In conclusion, by combining IF-3 chiral column and the multichannel-MRM method various molecular species of GSLs were detected successfully, and a metabolomics approach based on this LC-MS method should facilitate functional analysis of GSLs and the discovery of early biomarkers of glycosphingolipidoses at the molecular level.

Identification of Potent In Vivo Autotaxin Inhibitors that Bind to Both Hydrophobic Pockets and Channels in the Catalytic Domain.

Autotaxin (ATX, also known as ENPP2) is a predominant lysophosphatidic acid (LPA)-producing enzyme in the body, and LPA regulates various physiological functions, such as angiogenesis and wound healing, as well as pathological functions, including proliferation, metastasis, and fibrosis, via specific LPA receptors. Therefore, the ATX-LPA axis is a promising therapeutic target for dozens of diseases, including cancers, pulmonary and liver fibroses, and neuropathic pain. Previous structural studies revealed that the catalytic domain of ATX has a hydrophobic pocket and a hydrophobic channel; these serve to recognize the substrate, lysophosphatidylcholine (LPC), and deliver generated LPA to LPA receptors on the plasma membrane. Most reported ATX inhibitors bind to either the hydrophobic pocket or the hydrophobic channel. Herein, we present a unique ATX inhibitor that binds mainly to the hydrophobic pocket and also partly to the hydrophobic channel, inhibiting ATX activity with high potency and selectivity in vitro and in vivo. Notably, our inhibitor can rescue the cardia bifida (two hearts) phenotype in ATX-overexpressing zebrafish embryos.

Hexacosenoyl-CoA is the most abundant very long-chain acyl-CoA in ATP binding cassette transporter D1-deficient cells.

X-linked adrenoleukodystrophy (X-ALD) is an inherited disorder caused by deleterious mutations in the ABCD1 gene. The ABCD1 protein transports very long-chain FAs (VLCFAs) from the cytosol into the peroxisome where the VLCFAs are degraded through ß-oxidation. ABCD1 dysfunction leads to VLCFA accumulation in individuals with X-ALD. FAs are activated by esterification to CoA before metabolic utilization. However, the intracellular pools and metabolic profiles of individual acyl-CoA esters have not been fully analyzed. In this study, we profiled the acyl-CoA species in fibroblasts from X-ALD patients and in ABCD1-deficient HeLa cells. We found that hexacosenoyl (26:1)-CoA, but not hexacosanoyl (26:0)-CoA, was the most abundantly concentrated among the VLCFA-CoA species in these cells. We also show that 26:1-CoA is mainly synthesized from oleoyl-CoA, and the metabolic turnover rate of 26:1-CoA was almost identical to that of oleoyl-CoA in both WT and ABCD1-deficient HeLa cells. The findings of our study provide precise quantitative and metabolic information of each acyl-CoA species in living cells. Our results suggest that VLCFA is endogenously synthesized as VLCFA-CoA through a FA elongation pathway and is then efficiently converted to other metabolites, such as phospholipids, in the absence of ABCD1.

Complex formation of sphingomyelin synthase 1 with glucosylceramide synthase increases sphingomyelin and decreases glucosylceramide levels.

Sphingolipids, including sphingomyelin (SM) and glucosylceramide (GlcCer), are generated by the addition of a polar head group to ceramide (Cer). Sphingomyelin synthase 1 (SMS1) and glucosylceramide synthase (GCS) are key enzymes that catalyze the conversion of Cer to SM and GlcCer, respectively. GlcCer synthesis has been postulated to occur mainly in cis-Golgi, and SM synthesis is thought to occur in medial/trans-Golgi; however, SMS1 and GCS are known to partially co-localize in cisternae, especially in medial/trans-Golgi. Here, we report that SMS1 and GCS can form a heteromeric complex, in which the N terminus of SMS1 and the C terminus of GCS are in close proximity. Deletion of the N-terminal sterile α-motif of SMS1 reduced the stability of the SMS1-GCS complex, resulting in a significant reduction in SM synthesis in vivo In contrast, chemical-induced heterodimerization augmented SMS1 activity, depending on an increase in the amount and stability of the complex. Fusion of the SMS1 N terminus to the GCS C terminus via linkers of different lengths increased SM synthesis and decreased GlcCer synthesis in vivo These results suggest that formation of the SMS1-GCS heteromeric complex increases SM synthesis and decreases GlcCer synthesis. Importantly, this regulation of relative Cer levels by the SMS1-GCS complex was confirmed by CRISPR/Cas9-mediated knockout of SMS1 or GCS combined with pharmacological inhibition of Cer transport protein in HEK293T cells. Our findings suggest that complex formation between SMS1 and GCS is part of a critical mechanism controlling the metabolic fate of Cer in the Golgi.

Quantitative and Qualitative Method for Sphingomyelin by LC-MS Using Two Stable Isotopically Labeled Sphingomyelin Species.

We present a method of analyzing sphingomyelin (SM) qualitatively and quantitatively by liquid chromatography-electrospray Ionization-tandem mass spectrometry (LC-ESI-MS/MS). SM is a common sphingolipid composed of a phosphorylcholine and a ceramide as the hydrophilic and hydrophobic component, respectively. A number of SM species are present in mammalian cells due to a variety in the sphingoid long chain base (LCB) and an N-acyl moiety in the ceramide. In this report, we show a method of estimating the number of carbon and double bonds in a LCB and an N-acyl moiety based on their corresponding product ions in MS/MS/MS (MS3) experiments. In addition, we present a quantitative analysis method for SM using two stable isotopically labeled SM species, which facilitates determining the range used in SM quantitation. The present method will be useful in characterizing a variety of SM species in biological samples and industrial products such as cosmetics.

Profiling and Imaging of Phospholipids in Brains of Abcd1-Deficient Mice.

ABCD1 is a gene responsible for X-linked adrenoleukodystrophy (X-ALD), and is critical for the transport of very long-chain fatty acids (VLCFA) into peroxisomes and subsequent ß-oxidation. VLCFA-containing lipids accumulate in X-ALD patients, although the effect of ABCD1-deficiency on each lipid species in the central nervous system has not been fully characterized. In this study, each phospholipid and lysophospholipid species in Abcd1-deficient mice brains were profiled by liquid chromatography-mass spectrometry. Among the phospholipid and lysophospholipid species that are significantly more enriched in Abcd1-deficient mice brains, VLCFA were present in 75, 15, 5, 4, and 1 species of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, lysophosphatidylcholine, and lysophosphatidylethanolamine, respectively. Most VLCFA were incorporated at the sn-1 position of phosphatidylcholine and phosphatidylethanolamine. Among the phospholipid species that are significantly less enriched in Abcd1-deficient mice brains, odd-numbered saturated or mono-unsaturated fatty acyl moieties are contained in all phosphatidylcholine species. In addition, a number of phosphatidylglycerol, phosphatidylinositol, and phosphatidylserine species contained highly unsaturated fatty acyl moieties. Intriguingly, 44:1 phosphatidylcholine with VLCFA was mainly distributed in the gray matter, such as the cortex, but not in the white matter in the cerebrum and cerebellum. These results show that ABCD1-deficiency causes metabolic alternation of long-chain fatty acids and VLCFA. Moreover, our results imply a molecular mechanism for the incorporation of saturated or monounsaturated VLCFA into the sn-1 position of phospholipids, and also indicate that the distribution of phospholipids with VLCFA may correlate with the development of X-ALD.

Acyl Chain Preference in Foam Cell Formation from Mouse Peritoneal Macrophages.

Macrophage foam cells play critical roles in the initiation and development of atherosclerosis by synthesizing and accumulating cholesteryl ester (CE) in lipid droplets. However, in analyzing lipid metabolism in foam cell formation, studies have focused on the sterol group, and little research has been done on the acyl chains. Therefore, we adapted a model system using liposomes containing particular acyl chains and examined the effect of various acyl chains on foam cell formation. Of the phosphatidylserine (PS) liposomes tested containing PS, phosphatidylcholine, and cholesterol, we found that unsaturated (C18:1), but not saturated (C16:0 and C18:0), PS liposomes induced lipid droplet formation, indicating that foam cell formation depends on the nature of the acyl chain of the PS liposomes. Experiments on the uptake and accumulation of cholesterol from liposomes by adding [14C]cholesterol suggested that foam cell formation could be induced only when cholesterol was converted to CE in the case of C18:1 PS liposomes. Both microscopic observations and metabolic analysis suggest that cholesterol incorporated into either C16:0 or C18:0 PS liposomes may stay intact after being taken in by endosomes. The [14C]C18:1 fatty acyl chain in the C18:1 PS liposome was used to synthesize CE and triacylglycerol (TG). Interestingly, the [14C]C16:0 in the C18:1 PS liposome was metabolized to sphingomyelin rather than being incorporated into either CE or TG, which could be because of enzymatic acyl chain selectivity. In conclusion, our results indicate that the acyl chain preference of macrophages could have some impact on their progression to foam cells.

Comprehensive Quantitation Using Two Stable Isotopically Labeled Species and Direct Detection of N-Acyl Moiety of Sphingomyelin.

Sphingomyelin (ceramide-phosphocholine, CerPCho) is a common sphingolipid in mammalian cells and is composed of phosphorylcholine and ceramide as polar and hydrophobic components, respectively. In this study, a qualitative liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS/MS) analysis is proposed in which CerPCho structures were assigned based on product ion spectra corresponding to sphingosylphosphorylcholine and N-acyl moieties. From MS/MS/MS analysis of CerPCho, we observed product ion spectra of the N-acyl fatty acids as [RCO2]- ions as well as sphingosylphosphorylcholine. A calibration curve for CerPCho was constructed using two stable isotopically labeled CerPCho species and then used to quantify the CerPCho species in HeLa cells as a proof-of-principle study. The present study proposes an accurate method for quantifying and assigning structures to each CerPCho species in crude biologic samples by LC-ESI-MS/MS/MS analysis.

ATX-LPA1 axis contributes to proliferation of chondrocytes by regulating fibronectin assembly leading to proper cartilage formation.

The lipid mediator lysophosphatidic acid (LPA) signals via six distinct G protein-coupled receptors to mediate both unique and overlapping biological effects, including cell migration, proliferation and survival. LPA is produced extracellularly by autotaxin (ATX), a secreted lysophospholipase D, from lysophosphatidylcholine. ATX-LPA receptor signaling is essential for normal development and implicated in various (patho)physiological processes, but underlying mechanisms remain incompletely understood. Through gene targeting approaches in zebrafish and mice, we show here that loss of ATX-LPA1 signaling leads to disorganization of chondrocytes, causing severe defects in cartilage formation. Mechanistically, ATX-LPA1 signaling acts by promoting S-phase entry and cell proliferation of chondrocytes both in vitro and in vivo, at least in part through ß1-integrin translocation leading to fibronectin assembly and further extracellular matrix deposition; this in turn promotes chondrocyte-matrix adhesion and cell proliferation. Thus, the ATX-LPA1 axis is a key regulator of cartilage formation.

Overexpression of autotaxin, a lysophosphatidic acid-producing enzyme, enhances cardia bifida induced by hypo-sphingosine-1-phosphate signaling in zebrafish embryo.

Lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) are second-generation lysophospholipid mediators that exert multiple biological functions through their own cognate receptors. They are both present in the blood stream, activate receptors with similar structures (endothelial differentiation gene receptors), have similar roles in the vasculature and are vasoactive. However, it is unclear whether these lysophospholipid mediators cross-talk downstream of each receptor. Here, we provide in vivo evidence that LPA signaling counteracted S1P signaling. When autotaxin (Atx), an LPA-producing enzyme, was overexpressed in zebrafish embryos by injecting atx mRNA, the embryos showed cardia bifida, a phenotype induced by down-regulation of S1P signaling. A similar cardiac phenotype was not induced when catalytically inactive Atx was introduced. The cardiac phenotype was synergistically enhanced when antisense morpholino oligonucleotides (MO) against S1P receptor (s1pr2/mil) or S1P transporter (spns2) was introduced together with atx mRNA. The Atx-induced cardia bifida was prominently suppressed when embryos were treated with an lpar1 receptor antagonist, Ki16425, or with MO against lpar1. These results provide the first in vivo evidence of cross-talk between LPA and S1P signaling.

Molecular species of phospholipids with very long chain fatty acids in skin fibroblasts of Zellweger syndrome.

The ratio of C 26:0/C 22:0 fatty acids in patient lipids is widely accepted as a critical clinical criterion of peroxisomal diseases, such as Zellweger syndrome and X-linked adrenoleukodystrophy (X-ALD). However, phospholipid molecular species with very long chain fatty acids (VLCFA) have not been precisely characterized. In the present study, the structures of such molecules in fibroblasts of Zellweger syndrome and X-ALD were examined using LC-ESI-MS/MS analysis. In fibroblasts from Zellweger patients, a large number of VLCFA-containing molecular species were detected in several phospholipid classes as well as neutral lipids, including triacylglycerol and cholesteryl esters. Among these lipids, phosphatidylcholine showed the most diversity in the structures of VLCFA-containing molecular species. Some VLCFA possessed longer carbon chains and/or larger number of double bonds than C 26:0-fatty acid (FA). Similar VLCFA were also found in other phospholipid classes, such as phosphatidylethanolamine and phosphatidylserine. In addition, VLCFA-containing phospholipid species showed some differences among fibroblasts from Zellweger patients. It appears that phospholipids with VLCFA, with or without double bonds, as well as C 26:0-FA might affect cellular functions, thus leading to the pathogenesis of peroxisomal diseases, such as Zellweger syndrome and X-ALD.

Simultaneous quantitation of sphingoid bases and their phosphates in biological samples by liquid chromatography/electrospray ionization tandem mass spectrometry.

We developed a liquid chromatography/electrospray ionization tandem mass spectrometry method for the simultaneous quantitative determination of C18 sphingosine (Sph), C18 dihydrosphingosine (dhSph), C18 phytosphingosine (pSph), C18 sphingosine-1-phosphate (S1P), C18 dihydrosphingosine-1-phosphate (dhS1P), and C18 phytosphingosine-1-phosphate (pS1P). Samples were prepared by simple methanol deproteinization and analyzed in selected reaction monitoring modes. No peak tailing was observed on the chromatograms using a Capcell Pak ACR column (1.5 mm i.d. × 250 mm, 3 µm, Shiseido). The calibration curves of the sphingoids showed good linearity (r > 0.996) over the range of 0.050-5.00 pmol per injection. The accuracy and precision of this method were demonstrated using four representative biological samples (serum, brain, liver, and spleen) from mice that contained known amounts of the sphingoids. Samples of mice tissue such as plasma, brain, eye, testis, liver, kidney, lung, spleen, lymph node, and thymus were examined for their Sph, dhSph, pSph, S1P, dhS1P, and pS1P composition. The results confirmed the usefulness of this method for the physiological and pathological analysis of the composition of important sphingoids.